Compositions containing C-substituted diindolylmethane compounds

ABSTRACT

Disclosed are methods and compositions for the treatment of a wide array of cancers and tumors. In illustrative embodiments, diindolylmethanes, C-substituted diindolylmethanes, and analogs thereof have been described, which when administered either alone, or in combination with other anti-cancer or anti-tumorigenic compounds, provide new therapies for the treatment of cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional PatentApplications Serial Nos. 60/238,670 and 60/238,675, each filed on Oct.6, 2000.

FIELD OF THE INVENTION

[0002] The invention relates generally to methods and compositions forthe treatment of cancer. In particular, diindolylmethane, ringsubstituted diindolylmethane, C-substituted diindolylmethane, andanalogs thereof that possess potent antiestrogenic and antitumorigenicactivities are disclosed and used in anti-cancer applications.

BACKGROUND OF THE INVENTION

[0003] U.S. Pat. No. 5,516,790 (issued May 14, 1996) suggests a methodof inhibiting estrogen activity by administering a biologically activeamount of a substituted dibenzofuran or substituted dibenzodioxin.

[0004] U.S. Pat. No. 5,948,808 (issued Sep. 7, 1999) offers compoundsand compositions of substituted indole-3-carbinols and diindolylmethanesuitable for treating estrogen-dependent tumors along with methods oftreating such cancerous-conditions.

[0005] U.S. Pat. No. 6,136,845 (issued Oct. 24, 2000) suggests methodsand pharmaceutical combinations for inhibiting estrogen-dependent tumorsvia the co-administration of antiestrogen triphenylethylenes, includingtamoxifen and alkyl PCDFs.

[0006] Brockman, et al. (“Activation of PPARγ leads to inhibition ofanchorage independent growth of human colorectal cancer cells”Gastroenterology 115:1049-1055, 1998) suggests that PPAR agonists willbe effective antitumorigenic agents for treatment of colorectal cancer.

[0007] Chen, et al. (“Aryl Hydrocarbon receptor-mediated antiestrogenicand antitumorigenic activity of diindolylmethane,” Carcinogenesis,19:1631-1639, 1998) states that DIM represents a new class of relativelynon-toxic AhR-based antiestrogens that inhibit E2-dependent tumor growthin rodents.

[0008] Chen, et al. (“Indole-3-carbinol and diindolylmethane as arylhydrocarbon (Ah) receptor agonists and antagonists in T47D human breastcancer cells” Biochem. Pharmacol. 51:1069-1076, 1996) suggests that2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced CYP1A1-dependentethoxyresorufin O-deethylase (EROD) activity in human breast cells, andco-treatment with TCDD plus different concentrations of I3C or DIMresulted in a significant decrease in the induced response at thehighest concentration of I3C or DIM.

[0009] Duan, et al. (“Estrogen receptor-mediated activation of the serumresponse element in MCF-7 cells through MAPK-dependent phosphorylationof Elk-1” J. Biol. Chem. 276:11590-11598, 2001) suggests thattranscriptional activation of the serum response element by E2 was dueto ERalpha activation of the MAPK pathway and increased binding of theserum response factor and Elk-1 to the serum response element.

[0010] Elstner, et al. (“Ligands for peroxisome proliferator-activatedreceptor gamma and retinoic acid receptor inhibit growth and induceapoptosis of human breast cancer cells in vitro and in BNX mice” Proc.Natl. Acad Sci. USA 95:8806-8811, 1998) suggests that combinedadministration of troglitazone and all-trans-retinoic acid causesprominent apoptosis and fibrosis of MCF7 tumors in tripleimmunodeficient mice without toxic effects on the mice.

[0011] Garcia, et al. (“Constitutive activation of Stat3 by the Src andJAK tyrosine kinases participates in growth regulation of human breastcarcinoma cells” Oncogene 20:2499-2513, 2001) suggests that tyrosinekinases transduce signals through Stat3 protein that contribute to thegrowth and survival of human breast cancer cells in culture andpotentially in vivo.

[0012] Jeng, et al. (“Role of MAP kinase in the enhanced cellproliferation of long term estrogen deprived human breast cancer cells”Breast Cancer Res. Treat. 62:167-175, 2000) suggests that the MAP kinasepathway is, in part, involved in the adaptive process which results inenhanced DNA synthesis and cell proliferation in the absence ofexogenous estrogen in estradiol long term cells.

[0013] McDougal and Safe (“Methyl-substituted diindolylmethanes asAhR-based antitumorigenic/ antiestrogenic compounds” OrganohalogenCompounds, 37:253-256, 1998) suggests that methyl substituted DIMsinhibit estrogen induced breast cancer growth.

[0014] McDougal, et al. (“Inhibition of carcinogen-induced rat mammarytumor growth and other estrogen-dependent responses by symmetricaldihalo-substituted analogs of diindolylmethane” Cancer Letts.,151:169-179, 2000) suggests that dihalo-substituted analogs ofdiindolylmethane significantly inhibited mammary tumor growth while nosignificant changes in organ weights or liver and kidney histopathologywere observed.

[0015] Michaud, et al. (“Fruit and vegetable intake and incidence ofbladder cancer in a male prospective cohort” J. Natl. Cancer Inst.,91:605-613, 1999) suggests that high cruciferous vegetable consumptionmay reduce bladder cancer risk, but other vegetables and fruits may notconfer appreciable benefits against this cancer.

[0016] Mueller, et al. (“Terminal differentiation of human breast cancerthrough PPAR” Mol. Cell 1:465-470, 1998) suggests that the PPAR gammatranscriptional pathway can induce terminal differentiation of malignantbreast epithelial cells.

[0017] Ramamoorthy, et al. (“AhR-mediated antiestrogenicity ofdiindolylmethane and analogs in vivo and in vitro,” OrganohalogenCompounds, 37:321-324, 1998) suggests that DIM and substituted DIMsinhibit estrogen-induced uterine activities and breast cancer cellgrowth.

[0018] Safe (“2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and relatedenvironmental antiestrogens: characterization and mechanism of action,”in Endocrine Disrupters, Naz (ed.), CRC Press, Boca Raton, Fla., pp.187-221, 1999) suggests that selective Ah receptor modulators (SahRMs)are effective inhibitors of mammary tumor growth with clinical potentialfor treatment of breast cancer.

[0019] Suh, et al. (“A new ligand for the peroxisomeproliferator-activated receptor-PPAR, GW7845, inhibits rat mammarycarcinogenesis” Cancer Res. 59:5671-5673, 1999) suggests the use of aligand for peroxisome proliferator-activated receptor-gamma to preventexperimental breast cancer.

[0020] Tontonoz, et al. (“Terminal differentiation of human liposarcomacells induced by ligands for peroxisome proliferator-activated receptorand the retinoid X receptor” Proc. Natl. Acad. Sci. USA 94:237-241,1997) offers that PPAR gamma ligands such as thiazolidinediones andRXR-specific retinoids may be useful therapeutic agents for thetreatment of liposarcoma.

[0021] Zhou, et al. (“Inhibition of murine bladder tumorigenesis by soyisoflavones via alterations in the cell cycle, apoptosis andangiogenesis” Cancer Res., 58:5231-5238, 1998) suggests that soyisoflavones can inhibit bladder tumor growth through a combination ofdirect effects on tumor cells and indirect effects on the tumorneovasculature.

[0022] Cancer is one of the leading causes of premature death in mostdeveloped countries. Since 1990, more than five million people have diedfrom various forms of cancer. Presently, many cancer treatments areineffective, or display significant negative side effects. Thus, thereexists a need for the development of new and more effective treatmentsof cancer.

SUMMARY OF THE INVENTION

[0023] Previous studies have demonstrated that diindolylmethane (DIM)and related compounds inhibit mammary tumor growth in experimentalanimals and also inhibit mammary and endometrial cancer cellproliferation in various in vitro models (Chen et al., 1998). DIM isformed from indole-3-carbinol (I3C) in the gut, and I3C and relatedcompounds inhibit formation or growth of estrogen-regulated tumors inthe rodent mammary, endometrium and uterus, suggesting that thiscompound may be acting as an antiestrogen. Results of ongoing studieswith ring-substituted DIMs have demonstrated their potentantiestrogenic/antitumorigenic (patent submitted) activities. Researchin this laboratory has focused on the antiestrogenic activity of arylhydrocarbon receptor (AhR) agonists using2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as a model compound (Safe,1999). TCDD and related compounds inhibit mammary tumor growth in rodentmodels and 17β-estradiol (E2)-induced responses in the rodent uterus andhuman breast cancer cells. Subsequent research has shown that DIM andrelated compounds also bind the AhR, and mechanistic studies with DIMshow that the antiestrogenic and antitumorigenic activities are alsoAhR-mediated, although this does not exclude other mechanisms of action.

[0024] Several studies have indicated that the diet can influence theprocess of carcinogenesis, and both fruit and vegetables are reported topossess antimutagenic and anticarcinogenic properties in human, animaland cell models. A recent study investigated the role of fruit andvegetable intake on bladder cancer risk in a male prospective cohort of252 bladder cancer cases among 47,909 men enrolled in the HealthProfessionals Follow-up Study (Michaud et al., “Fruit and vegetableintake and incidence of bladder cancer in a male prospective cohort” J.Natl. Cancer Inst., 91:605-613, 1999). A detailed analysis showed thatthere was a significant correlation between decreased bladder cancerrisk and increased dietary intake of cruciferous vegetables suggestingthat high cruciferous vegetable consumption may reduce bladder cancerrisk. It was suggested that compounds such as the isothiocyanatesulforaphane (Michaud et al., “Fruit and vegetable intake and incidenceof bladder cancer in a male prospective cohort” J. Natl. Cancer Inst.,91:605-613, 1999) that induce phase 2 drug metabolizing enzymes may bechemoprotective.

[0025] Cruciferous vegetables including broccoli, cauliflower, Brusselssprouts, and cabbage contain several compounds such as indoles,isothiocyanates and dithiolthiones which modulate carcinogenesis indifferent animal models. For example, glucobrassicin (3-indolylmethylglucosinolate), a major component of cauliflower (0.1 to 1.6 mmol/kg),cabbage (0.1 to 1.9 mmol/kg), and Brussels sprouts (0.5 to 3.2 mmol/kg)is readily converted to indole-3-carbinol (I3C). Therefore, it appearsthat DIM and related compounds may be not only chemopreventative, butalso act as antitumorigenic agents for bladder and other cancers throughthe AhR and possibly other mechanistic pathways.

DESCRIPTION OF THE FIGURES

[0026] The following figures form part of the present specification andare included to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein. FIGURE Description 1 Illustratesinduced transformation of the rat hepatic cytosolic AhR and binding to[³²P]DRE in a gel mobility shift assays as described (Chen et al.,1998). Cytosol was treated with DMSO (lane 1), TCDD (lane 2), TCDD plusexcess DRE (lane 3) or mutant DRE (lane 4). Lanes 5-13 were treated withthe following compounds substituted at R₈ (note: R₁/R_(1′) = CH₃ or H):R₁/R_(1′) = CH₃, R₈ = p-C₆H₄Cl; R₁/R_(1′), R₈ = C₆H₄—C₆H₅; R₁/R_(1′), R₈= C₁₀H₇ (naphthyl); R₈ = pC₆H₄OH; R₁/R_(1′) = CH₃, R₈ = C₆H₅OCH₃;R₁/R_(1′) = CH₃, R₈ = pC₆H₄OH; R₈ = pC₆H₅OCH₃; R₈ = C₆H₄—C₆H₅. 2Illustrates the effects of C—PhDIM (R₈ = C₆H₅) and C—MeDIM (R₈ = Me)alone and in combination with 1 nM E2 on proliferation of MCF-7 humanbreast cancer cells. 3 Illustrates estrogenic and antiestrogenicactivities of substitutes DIMs in T47D cells. Effects of differentconcentrations of substitutes DIMs alone or in combination with 1 nM E2on cell proliferation was determined as described in Materials andMethods. Results are expressed as means ± SE for at least threereplicate experiments for each treatment group. *Significant inhibition(p < 0.05) of E2-induced cell proliferation. The compounds used in thisstudy include C—MeDIM (R₈ = CH₃), C—PhDIM (R₈ = C₆H₅), Nme—C—Ph—OHDIM(R₁/R_(1′) = CH₃; R₈ = p-C₆H₄OH), and NMe— CPh—MeODIM (R₁/R_(1′) = CH₃;R₈ = p-C₆H₄OCH₃). 4 Illustrates inhibition of DMBA-induced mammary tumorgrowth in female Sprague-Dawley rats. Rats were treated with C—PhDIM (R₈= C₆H₅) (1.0 mg/kg/2 d) and tumor volumes in treated and control (cornoil) animals determined over a period of 21 days as described (McDouglaset al., 2000). 5 Illustrates inhibition of DMBA-induced mammary tumorgrowth in female Sprague-Dawley rats. Rats were treated with NMe—C—PhDIM(R₁/R_(1′) = CH₃; R₈ = C₆H₅) (1.0 mg/kg/2 d) and tumor volumes intreated and control (corn oil) animals determined over a period of 21days as described (McDouglas et al., 2000). 6 Illustrates inhibition ofDMBA-induced mammary tumor growth in female and C—Ph—CF₃DIM (R₈ =p-C₆H₄—CF₃) (1.0 mg/kg/2 d) and tumor volumes in treated and control(corn oil) animals determined over a period of 21 days as described(McDouglas et al., 2000). 7 Illustrates inhibition of DMBA-inducedmammary tumor growth in female Sprague-Dawley rats. Rats were treatedwith NMe—C—Ph—MeODIM (R₁/R_(1′) = CH₃; R₈ = C₆H₄OCH₃), NMe—C—Ph—OHDIM(R₁/R_(1′) = CH₃; R₈ = C₆H₄OH), and NMe—C-NaphthylDIM (R₁/R_(1′) = CH₃;R₈ = C₁₀H₇) (1.0 mg/kg/2 d) and tumor volumes in treated and control(corn oil) animals determined over a period of 21 days as described(McDouglas et al., 2000). 8 Illustrates inhibition of DMBA-inducedmammary tumor growth in female Sprague-Dawley rats. Rats were treatedwith NMe—BiPhDIM (R₁/R_(1′) = CH₃; R₈ = C₆H₄C₆H₅), NMe—C—Ph—MeDIM(R₁/R_(1′) = CH₃; R₈ = p- C₆H₄CH₃), and NMe—C—Ph—CF₃DIM (R/R_(1′) = CH₃;R₈ = p-C₆H₄CF₃) (1.0 mg/kg/2 d) and tumor volumes in treated and control(corn oil) animals determined over a period of 21 days as described(McDouglas et al., 2000). 9 Illustrates comparative inhibition of TCCSUPbladder cancer cell growth by 0.1-50 μM genistein and DIM. 10 DepictsC-substituted diindolylmethane (DIM) compounds. 11 Depicts the overallscheme for the synthesis of C-substituted DIMs. 12 Induction of ERODactivity of TCDD or DIM in PC3 prostate cancer cells: effects ofconcentration and treatment time. 13 Induction of EROD activity in22Rv1prostate cancer cells after treatment with DIM or TCDD for 24 hrs.14 Induction of luciferase activity by 5: M C-substituted DIMs and PG-J2in MCF-7 breast cancer cells transfected with pGAL4₅ and pGAL4-PPAR(. 15Induction of luciferase activity in cells transfected with pSRE, PMAPKK,and treated with DIMs. 16 Inhibition of 22Rv1 prostrate cancer cellgrowth by TCDD and DIM (6 days of growth in media containing 1% serum).17 Inhibition of PC3 prostrate cancer cell growth by DIM in cells grownin 1% serum for 6 days.

DEFINITIONS

[0027] The following definitions are provided in order to aid thoseskilled in the art in understanding the detailed description of thepresent invention.

[0028] “AhR” refers to aryl hydrocarbon receptor.

[0029] “DIM” refers to diindolylmethane.

[0030] “DMBA” refers to 7,12-dimethylbenz[a]anthracene.

[0031] “ER” refers to estrogen receptor.

[0032] “EROD” refers to ethoxyresorufin O-deethylase.

[0033] “E2” refers to 17β-estradiol.

[0034] “I3C” refers to indole-3-carbinol.

[0035] “MAPKK” refers to mitogen-activated protein kinase

[0036] “PPARY” refers to peroxisome proliferator activity receptor y.

[0037] “PCDF” refers to polycholorinated dibenzofuran.

[0038] “TCDD” refers to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

DETAILED DESCRIPTION OF THE INVENTION

[0039] Provided in the present invention is a compound having thestructure:

[0040] where R₁, R₂, R₄, R₅, R₆, R₇, R₁′, R₂′, R4′, R₅′, R₆′, and R₇ ′independently, is hydrogen, or a substituent selected from the groupconsisting of a halogen, a nitro group, and a linear or branched alkylor alkoxy group of about one to about ten carbons, preferably of aboutone to about five carbons, said compound having at least onesubstituent. The halogen is selected from the group consisting ofchlorine, bromine, and fluorine. A compound such as this is referred toas a DIM derivative or a DIM analog.

[0041] In a preferred embodiment of the DIM derivatives, R₁, R₂, R₄, R₆,R₇, R₁′, R₂′, R₄′, R₆′, and R₇′are hydrogen, R₅ and R₅′ are a halogenselected from the group consisting of chlorine, bromine and fluorine.Accordingly, preferred DIM derivatives include5,5′-dichloro-diindolylmethane, 5,5′-dibromo-diindolylmethane, and5,5′-difluoro-diindolylmethane.

[0042] Additional preferred DIM derivatives include compounds whereinR₁, R₂, R₄, R₆, R₇, R₁′, R₂′, R₄′, R₆′, and R₇′ are hydrogen, R₅ and R₅′are an alkyl or alkoxyl having from one to ten carbons, and mostpreferably one to five carbons. These include, but are not limited to5,5′-dimethyl-diindolylmethane, 5,5′-diethyl-diindolylmethane,5,5′-dipropyl-diindolylmethane, 5,5′-dibutyl-diindolylmethane and5,5′-dipentyl-diindolylmethane. These also include, but are not limitedto, 5,5′-dimethoxy-diindolylmethane, 5,5′-diethoxy-diindolylmethane,5,5′-dipropyloxy-diindolylmethane, 5,5′-dibutyloxy-diindolylmethane, and5,5′-diamyloxy-diindolylmethane.

[0043] Additional preferred DIM derivatives include compounds whereinR₂, R₄, R₅, R₆, R₇, R₂′, R₄′, R₅′, R₆′, and R₇′ are hydrogen, R₁andR₁′are an alkyl or alkoxyl having from one to ten carbons, and mostpreferably one to five carbons. Such useful derivatives include, but arenot limited to, N,N′-dimethyl-diindolylmethane,N,N′-diethyl-diindolylmethane, N,N′-dipropyl-diindolylmethane,N,N′-dibutyl-diindolylmethane, and N,N′-dipentyl-diindolylmethane.

[0044] In yet another preferred embodiment, R₁, R₄, R₅, R₆, R₇, R₁′,R₄′, R₅′, R₆′, and R₇′ are hydrogen, and R₂ and R₂′ are alkyl of one toten carbons, and most preferably one to about five carbons. Suchcompounds include, but are not limited to,2,2′-dimethyl-diindolylmethane, 2,2′-diethyl-diindolylmethane,2,2′-dipropyl-diindolylmethane, 2,2′-dibutyl-diindolylmethane, and2,2′-dipentyl-diindolylmethane.

[0045] In another embodiment, R₁, R₂, R₄, R₆, R₇, R₁′, R₂′, R₄′, R₆′,and R₇ ′are hydrogen, and R₅ and R₅′ are nitro.

[0046] An alternative embodiment of the invention is directed towardsDIM compounds with modifications at the bridge carbon (“C-substitutedDIMs”). These compounds can be symmetrical or asymmetrical, depending onwhether a single indole precursor is used in the synthesis (leading to asymmetrical C-substituted DIM, or if two different indole precursorswere used (leading to an asymmetrical C-substituted DIM). TheC-substituted DIMs are generally represented by the following structure:

[0047] The scope of possible substituents at the R₁ through R₇ (and R₁′through R₇′) are the same as described above in relation to DIMs. R₈ andR₈′ are each independently selected from the group consisting ofhydrogen, a linear alkyl group containing one to about ten carbon atoms,a branched alkyl group containing one to about ten carbon atoms, acycloalkyl group containing one to about ten carbon atoms, and an arylgroup. At least one of R₈ and R₈′ are not hydrogen (if both R₈ and R8′are hydrogen, the compound is a DIM). A preferred embodiment ofC-substituted DIMs includes when R₁, R₂, R₁′, and R₂′ are eachindividually hydrogen or methyl; R₄, R₅, R₆, R₇, R₄′, R₅′, R₆and R₇′ areeach hydrogen; and R₈ and R₈′ are each individually hydrogen, methyl,C₆H₅, C₆H₄OH, C₆H₄CH₃, C₆H₄CF₃, C₁₀H₇, C₆H₄C₆H₅, or C₆H₄OCH₃. Dependingon the nature of the two indole subunits, and of R₈ and R₈′, it ispossible for the bridging carbon atom to be a chiral center (a carbonatom with four different substituents attached). If a chiral centerexists, then the resulting C-substituted DIM would consist of two mirrorimage enantiomers, each of which is optically active. Resolution of themixture using a chiral chromatography column or other means would resultin the isolation of purified or pure enantiomer products. The differentenantiomers may prove to have different biological activities.

[0048] The synthesis of the substituted I3C derivatives from thecommercially-available substituted indoles is a convenient method forpreparation of these compounds. The substituted DIM analogs can also beprepared by condensation of formaldehyde with substituted indoles;however, a disadvantage of the latter reaction is the formation ofby-products which will complicate purification of the desiredsubstituted DIM. The compounds of the present invention can besynthesized by dimethylformamide condensation of a suitable substitutedindole to form a substituted indole-3-carboxaldehyde. Suitablesubstituted indoles include those indoles having substituents at R₁, R₂,R₄, R₅, R₆ and R₇ positions. These include, but are not limited to5-methoxy, 5-chloro,5-bromo, 5-fluoro, 5-methyl, 5-nitro, N-methyl, and2-methyl indoles. The substituted indole 3-aldehyde product is treatedwith a suitable alcohol such a methanol and solid sodium borohydride toreduce the aldehyde moiety to give substituted I3Cs. Substituted DIMsare prepared by condensing the substituted indole-3-carbinol products.This may be achieved, for example, by treatment with a phosphate bufferhaving a pH of about 5.5. Use of a single indole starting material willlead to symmetrical products, while use of two different indole startingmaterials will lead to asymmetrical products.

[0049] The agents of the present invention may be administeredtopically, orally, by injection (IV, IP, IM), intranasally,transdermally, rectally, or by any means which delivers an effectiveamount of the active agent to the tissue or site to be treated. Suitabledosages are those which achieve the desired endpoint. It will beappreciated that different dosages may be required for treatingdifferent disorders. An effective amount of an agent is that amountwhich causes a significant decrease in neoplastic cell count, growth, orsize.

[0050] Any of the above-described compounds can be used to treat cancer,either in vitro or in vivo. The cancer is generally any type of cancer,preferably adrenal cortical cancer, anal cancer, bile duct cancer, bonecancer, bone metastasis, brain cancer, cervical cancer, non-Hodgkin'slymphoma, rectum cancer, esophageal cancer, eye cancer, gallbladdercancer, gastrointestinal carcinoid tumors, gestational trophoblasticdisease, Hodgkin's disease, Kaposi's sarcoma, kidney cancer, laryngealand hypopharyngeal cancer, leukemia, liver cancer, lung cancer, lungcarcinoid tumors, malignant mesothelioma, metastatic cancer, multiplemyeloma, myelodysplastic syndrome, nasal cavity and paranasal cancer,nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngealcancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer,pituitary cancer, retinoblastoma, salivary gland cancer, sarcoma, skincancer, stomach cancer, testicular cancer, thymus cancer, thyroidcancer, uterine sarcoma, vaginal cancer, vulva cancer, Wilm's tumor andmore preferably is bladder, colon or prostate cancer.

[0051] Those having ordinary skill in the art will be able to ascertainthe most effective dose and times for administering the agents of thepresent invention, considering route of delivery, metabolism of thecompound, and other pharmacokinetic parameters such as volume ofdistribution, clearance, age of the subject, and so on.

[0052] The active agents may be administered along with a pharmaceuticalcarrier and/or diluent. The agents of the present invention may also beadministered in combination with other agents, for example, inassociation with other chemotherapeutic or immunostimulating drugs ortherapeutic agents. Examples of pharmaceutical carriers or diluentsuseful in the present invention include any physiological bufferedmedium, i.e., about pH 7.0 to 7.4 comprising a suitable water solubleorganic carrier. Suitable water soluble organic carriers include, butare not limited to corn oil, dimethylsulfoxide, gelatin capsules, and soon.

[0053] The present invention is exemplified in terms of in vitro and invivo activity against various neoplastic and normal cell lines. The testcell lines employed in the in vitro assays are well recognized andaccepted as models for antitumor activity in animals. The term “animals”as used herein includes, but is not limited to, mice, rats, domesticatedanimals such as, but not limited to cats and dogs, and other animalssuch as, but not limited to cattle, sheep, pigs, horses, and primatessuch as, but not limited to monkeys and humans. The mouse experimentaltumor in vivo assays are also well recognized and accepted as predictiveof in vivo activity in other animals such as, but not limited to,humans.

[0054] The following examples are included to demonstrate preferredembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventors to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

EXAMPLES Example 1 Synthesis of Diindolylmethane

[0055] Indole or ring-substituted indoles (e.g., 5-methoxy, 5-chloro,5-bromo, 5-fluoro, 5-methyl, 5-nitro, N-methyl and 2-methyl) arecommercially available and these compounds are used for synthesis ofdiindolylmethane analogs. Alkyl, substituted alkyl, aromatic, orsubstituted aromatic aldehydes (0.01 mole) are incubated with indole ora substituted indole (0.02 mole) in water (50 ml) plus glacial aceticacid (0.5 ml). Depending on the structure of the aldehyde or indole, thereaction is continued with stirring for 2 days to 2 weeks. The reactionproduct is either filtered or isolated by extraction with chloroform andthe residue crystallized from benzene/petroleum spirit. The resultingsubstituted DIM is then used in in vivo or in vitro studies. DIMs tendto be photosensitive and should be stored in dark brown vials.

Example 2 Diindolylmethane Analogs as Antitumorigenic Agents for theTreatment of Multiple Cancers

[0056] Initial studies used TCCSUP and J82 human bladder cancer celllines that were maintained in RPMI culture media supplemented with 10%fetal bovine serum. Cell proliferation studies were carried out inmulti-well plates, and compounds (in DMSO, 0.1% vol./vol.) were addedand cell growth was determined over a period of 4-10 days. The growthinhibitory properties of DIM and related analogs were determined usingthe following prototypical compounds: DIM, 5,5′-dimethylDIM (5-Me-DIM),2,2′-dimethylDIM (2-Me-DIM), and 5,5′-dichloroDIM (5-Cl-DIM) atconcentrations from 0.1-10 μM. Proliferation of both human bladdercancer cell lines was significantly inhibited by all compounds used inthis study; moreover, the more sensitive TCCSUP cells were inhibited byall compounds at the lowest concentration (0.1 μM) (FIG. 1).Interestingly, the DIM analogs were>100 times more inhibitory thangenistein and related bioflavonoids in soy products that inhibitedgrowth of these bladder cancer cells at 10 μM concentrations. Theseresults suggest that DIM analogs exhibit excellent potential asinhibitors of bladder cancer since the soy products were also active asin vivo inhibitors of bladder cancer tumor growth.

[0057] Studies with HT-29 human colon cancer cells also indicate thatDIM and related analogs protect against colon cancer. HT-29 cells weregrown in DMEM and serum and treated with 25 μM DIM and 4,4′-dichloroDIM.After 72 hr, there was a 73 and 92% decrease in cell proliferation,respectively, and these antiproliferative effects could be observed withcell concentrations as low as 10 μM. These growth inhibitory effectswere also accompanied by programmed cell death or apoptosis.

Example 3 PPAR(Activity of DIM Analogs

[0058] Peroxisome proliferator-activated receptor (PPAR) is a nuclearreceptor that induces differentiation in multiple tissues and celllines. Synthetic compounds that bind PPAR(inhibit growth, inducedifferentiation in multiple tumor cell lines, and inhibit mammarycarcinogenesis in rodent models (Mueller et al., “Terminaldifferentiation of human breast cancer through PPAR” Mol. Cell1:465-470, 1998; Elstner et al., “Ligands for peroxisomeproliferator-activated receptor gamma and retinoic acid receptor inhibitgrowth and induce apoptosis of human breast cancer cells in vitro and inBNX mice” Proc. Natl. Acad. Sci. USA 95:8806-8811, 1998; Brockman etal., “Activation of PpaRg leads to inhibition of anchorage independentgrowth of human colorectal cancer cells” Gastroenterology 115:2049-1055,1998; Tontonoz et al., “Terminal differentiation of human lipsarcomacells induced by ligands for peroxisome proliferator-activated receptorand the retinoid X receptor” Proc. Natl. Acad. Sci. USA 94:237-241,1997; Suh et al., “A new ligand for the peroxisomeproliferator-activated receptor-PPAR, GW7845, inhibits rat mammarycarcinogenesis” Cancer Res. 59:5671-5673, 1999). We have beeninvestigating the potential role of DIMs as ligands for the PPARγreceptor using a chimeric protein containing the yeast GAL4 DNA bindingdomain fused to the PPARγ ligand binding domain (pGAL4-PPARγ). Ligandactivation of pGAL4-PPARγ is detected using a construct containing fivetandem GAL4 response elements (pGAL4₅) linked to a luciferase reportergene. The results illustrated in FIG. 14 show that 5 μM15-deoxy-γ^(12,14)-prostaglandin J₂ (PGJ) (a prototypical ligand forPPARγ induced a 2-fold increase in reporter gene activity in MCF-7breast cancer cells, and similar results were obtained with 5 μMconcentrations of DIM and 1,1′-dimethylDIM analogs containingC-substituted p-trifluoromethylphenyl substituents (FIG. 14). Thissuggests that substituted DIMs may also inhibit cancer growth throughbinding and activation of PPARγ, and we are currently investigatingactivities of other analogs in binding and functional assays.

Example 4 DIMs as Kinase Inhibitors

[0059] Many tumors overexpress growth factor receptors and othermembrane receptors and exhibit activated kinase activities whichcontribute to the high rate of tumor growth (Garcia et al.,“Constitutive activation of Stat3 by the Src and JAK tyrosine kinasesparticipates in growth regulation of human breast carcinoma cells”Oncogene 20:2499-2513, 2001; Jeng et al., “Role of MAP kinase in theenhanced cell proliferation of long term estrogen deprived human breastcancer cells” Breast Cancer Res. Treat. 62:167-175, 2000). Therefore, wehave been investigating the inhibitory effects of DIMs on kinaseactivities and our initial studies have examined inhibition ofmitogen-activated protein kinase (MAPKK) which regulates expression ofmultiple genes involved in cell proliferation. MCF-7 cells aretransfected with a constitutively active MAPKK expression plasmid(pMAPKK), and induction of activity is monitored through activation ofthe construct pSRE which contains a MAPKK-inducible serum responseelement (SRE) from the cfos protooncogene (Duan et al., “Estrogenreceptor-mediated activation of the serum response element in MCF-7cells through MAPK-dependent phosphorylation of Elk-I” J. Biol. Chem.276:11590-11598, 2001). The results (FIG. 15) indicate that 1 or 5 μMconcentrations of 5,5′- and 6,6′-dimethlyDIM inhibited MAPKK-inducedactivity suggesting that DIMs can also exhibit growth inhibitoryproperties in cancer cells by direct inhibition of kinase activity, andwe are currently investigating inhibition of other growth relatedkinases by DIMs.

Example 5 Inhibition of Prostate Cancer Cell Growth

[0060] We examined the dose-dependent effects of DIM on proliferation ofPC3 human prostate cancer cells in culture, and the results (FIG. 16)showed that in DIM (100 nM -10 μM) significantly inhibited growth ofthis cell lines in 1% serum. In a second study using 22Rv1 humanprostate cancer cells, both 1 nM TCDD (an Ah receptor ligand) and 10 μMDIM inhibited proliferation of this cell line, thus confirming theanticancer activity of DIMs on prostate cancer cells.

Example 6 Synthesis of C-Substituted DIMs

[0061] The overall scheme for synthesis of C-substituted DIMs is shownin FIG. 11. A substituted indole (10 mmol) containing one or moresubstituents (R₁-R₇) is incubated with a slight molar excess of asubstituted aldehyde (R8-CHO, 10 mmol) or ketone in 50 ml water and 0.6ml glacial acetic acid. The mixture is stirred rapidly for 1-30 days,and the formation of DIM condensation product is monitored by gas-liquidor thin-layer chromatography. After the reaction is complete, thecondensation mixture is filtered, washed with distilled water, dried andcrystallized from benzene or benzene/petroleum spirit to give a purecondensation product. The reaction products may be light sensitive andshould be synthesized and stored in the dark. In addition, unsymmetricalcondensation products using different substituted indoles can also besynthesized, purified and separated by high performance liquidchromatography for biological screening. In addition, symmetrical orunsymmetrical ketones (R₈, R₈′ C=O) can be used to give additionalsubstituted DIMs at the bridged carbon atoms.

[0062] C-substituted diindolylmethanes (DIMs) include the generalizedset of compounds shown in FIG. 10, where R₈/R₈′ are substituents at theC-bridge, and R₁/R₁′ -R₇/R₇′ are substituents at positions 1, 2, 4, 5, 6and 7. Table 1 illustrates a range of prepared compounds. TABLE 1 R₁ H,H, H, H, H, H, H, H, H, CH₃ CH₃ CH₃ CH₃ CH₃ CH₃ CH₃ CH₃ CH₃ R_(2,4-7) HH H H H H H H 2-CH₃ R₈ CH₃ C₆H₅ C₆H₄— C₆H₄— C₆H₄— C₁₀H₇ C₆H₄— C₆H₄— CH₃OH CH₃ CF₃ C₆H₅ OCH₃

[0063] All possible substituents on the ring (i.e., more than oneR_(1,2,4-7) as well as different substituents on both rings, i.e.,unsymmetrical substitution) and at the bridge C-atom [i.e., one or twobridge substituents (R₈/R_(8′))] that may be the same or different.

Example 7 AhR Activities

[0064] The DIM series of compounds containing both ring and methylene -Csubstituents can be used for treating multiple cancers through both Ahreceptor-dependent and-independent pathways. Many of these compoundsbind the Ah receptor; however, it is suspected that they may alsoinhibit tumor growth by other mechanisms, such as through activation ofPPARγ (Example 3). Results illustrated below summarize theconcentration-dependent induction of CYP1A1-dependent ethoxyresorufinO-deethylase (EROD) activity by DIM and TCDD in androgen-nonresponsivePC3 human prostate cancer cells (FIG. 12). Initial studies showed thatminimal (but significant) induction was observed after 24 hours;however, 10 μM DIM and 10 nM TCDD induced EROD activity which wasmaximal (for TCDD) after treatment for 96 hours. The fold-inductionresponse for DIM was lower than observed for TCDD even at concentrationsof DIM that were 1000 times higher than TCDD, and this response istypical for SahRMs such as DIM which exhibit low Ah receptor-mediatedtoxicities (Chen et al., “Indole-3-carbinol and diindolylmethane as arylhydrocarbon (Ah) receptor agonists and antagonists in T47D human breastcancer cells” Biochem. Pharmacol 51:1069-1076, 1996). We alsoinvestigated the induction of EROD activity in two additionalandrogen-responsive prostate cancer cell lines. The results illustratedin FIG. 13 show that 0.1 to 10 nM TCDD induced EROD activity inandrogen-responsive 22 Rv1 prostate cancer cells (top), and DIM alsoinduced a minimal (but significant) increase in EROD activity (middle).In combination studies, higher concentration of DIM inhibited TCDDinduced activity, and this is consistent with results of previousstudies which show that DIM interacts directly with CYP1A1 protein andinhibits catalytic activity such as EROD (Chen et al.,“Indole-3-carbinol and diindolylmethane as aryl hydrocarbon (Ah)receptor agonists and antagonists in T47D human breast cancer cells”Biochem. Pharmacol. 51:1069-1076, 1996). We have also investigated theinduction of EROD activity by TCDD in androgen-responsive LnCAP prostatecancer cells and there was also significant induction of EROD activity.Thus, human prostate cancer cells express a functional Ah receptor.

[0065] Studies have demonstrated that DIM and ring-substituted DIManalogs exhibit antiestrogenic activities in breast cancer cells andantitumorigenic activity in the carcinogen-induced rat mammary tumormodel (Chen et al., “Aryl Hydrocarbon receptor-mediated antiestrogenicand antitumorigenic activity of diindolylmethane,” Carcinogenesis,19:1631-1639, 1998; McDougal et al., “Inhibition of carcinogen-inducedrat mammary tumor growth and other estrogen-dependent responses bysymmetrical dihalo-substituted analogs of diindolylmethane” CancerLetts., 151:169-179, 2000). However, the activities of the C-substitutedanalogs have not been determined. Moreover, it might be expected thatC-substitution with alkyl, phenyl or other aromatic substituents woulddecrease binding affinity to the Ah receptor and render these compoundsinactive as Ah receptor based antiestrogens. Results of preliminarystudies with some C-substituted DIMs indicated that most of thesecompounds only weakly induce transformation of the rat cytosolic Ahreceptor suggesting that they exhibit some Ah receptor-like activity(FIG. 1).

[0066] Studies indicate that a few of the substituted DIMs alsotranscriptionally activate peroxisome proliferator activity receptor γ(PPARγ) (Example 3) and some PPARγ agonists also inhibitcarcinogen-induced mammary tumor growth.

Example 8 In Vitro Studies with Breast Cancer Cells

[0067] Previous studies have demonstrated that DIM and substituted DIMsinhibited estrogen-induced growth of breast cancer cells in culture(Chen et al., “Aryl Hydrocarbon receptor-mediated antiestrogenic andantitumorigenic activity of diindolylmethane,” Carcinogenesis,19:1631-1639, 1998; McDougal et al., “Inhibition of carcinogen-inducedrat mammary tumor growth and other estrogen-dependent responses bysymmetrical dihalo-substituted analogs of diindolylmethane” CancerLetts., 151:169-179, 2000) and these studies have also been carried outwith C-substituted DIMs. The results in FIG. 2 summarize theantiestrogenic activity of two C-substituted DIMs wherein R₈=CH₃(methyl) or C₆H₅ (phenyl) and R₁-R₈=H. The results show that atconcentrations up to 10 μM, the compounds alone do not affect growth ofMCF-7 breast cancer cell lines. However, in cells co-treated with 1 nMestradiol (E2) plus different concentrations of the C-CH₃ or C-C₆H₅substituted DIMs, there was significant inhibition of E2-induced cellproliferation. In a separate study, it was also shown that the samecompounds and other C-substituted DIMs were also antiestrogenic in T47Dcells, and these include the C-p-phenol and C-p-anisole compounds inwhich R₁=CH₃ (methyl) (i.e., NMe-CPh-OHDIM and NMe-CPh-MeODIM,respectively) (FIG. 3).

Example 9 In Vivo Antitumorigenic Activity of C-Phenyl Substituted DIMin the DMBA-Induced Rat Mammary Tumor Model

[0068] Several studies have previously demonstrated that AhR agonistsexhibit antiestrogenic activity in both in vivo and in vitro models(Chen et al.,“Aryl Hydrocarbon receptor-mediated antiestrogenic andantitumorigenic activity of diindolylmethane,” Carcinogenesis,19:1631-1639, 1998; McDougal et al.,“Inhibition of carcinogen-inducedrat mammary tumor growth and other estrogen-dependent responses bysymmetrical dihalo-substituted analogs of diindolylmethane” CancerLetts., 151:169-179, 2000). Research has identified a series ofalternate substituted alkyl polychlorinated dibenzofurans (PCDFs) thatbind to the AhR, exhibit low toxicity but are relatively potentantiestrogens in both in vivo and in vitro studies(Safe,“2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and relatedenvironmental antiestrogens: characterization and mechanism of action,”in Endocrine Disrupters, Naz (ed.), CRC Press, Boca Raton, FL, pp.187-221, 1999). These compounds inhibit mammary tumor growth in femaleSprague-Dawley rats initiated with 7,12-dimethylben[a]anthracene (DMBA)and the antitumorigenic activities of alkyl PCDFs are not accompanied byany apparent liver or extrahepatic toxic effects. Comparable studieshave been carried out using DIM and ring-substituted DIMs, and thesecompounds are also relatively nontoxic Ah receptor-based antiestrogensthat block mammary tumor growth in vivo (Chen et al.,“Aryl Hydrocarbonreceptor-mediated antiestrogenic and antitumorigenic activity ofdiindolylmethane,” Carcinogenesis, 19:1631-1639, 1998; McDougal etal.,“Inhibition of carcinogen-induced rat mammary tumor growth and otherestrogen-dependent responses by symmetrical dihalo-substituted analogsof diindolylmethane” Cancer Letts., 151:169-179, 2000).

[0069]FIG. 4 illustrates the potent inhibition of mammary tumor growthby C-PhDIM (R₈=C₆H₅) in DMBA-induced Sprague-Dawley rats. Theanticarcinogenic response was observed at a dose of 1.0 mg/kg every 2days and as shown in Table 2, this was not accompanied by changes inbody/organ weights or altered tissue histopathology. TABLE 2Antitumorigenic activity of C-PhenylDIM in female Sprague-Dawley RatsControl C-PhenylDIM Tumor volume (mm³) 3332 ± 701  1025 ± 245* Tumorweight (g) 5.90 ± 2.0  1.40 ± 0.4* EROD (pmol/mg/min) 448 ± 170 309 ±102 Liver (% body weight) 3.1 ± 0.2 3.2 ± 0.1 Uterus (% body weight)0.15 ± 0.02 0.19 ± 0.02 Heart (% body weight) 0.25 ± 0.01 0.38 ± 0.03Spleen (% body weight) 0.30 ± 0.03 0.33 ± 0.05 Kidney (% body weight)0.32 ± 0.01 0.33 ± 0.01

[0070] Similar results have been observed for other C-substituted DIMsincluding NMe-C-PhDIM (R₁/R_(1′)=CH₃; R₈=C₆H₅; other R groups=H) (FIG.5); C-BiphDIM (R₈=C₆H₄-C₆H₅; all other R groups=H) (FIG. 6);NMe-C-Ph-MeODIM (R₁/R_(1′)=CH₃; R₈=C₆H₄-OH; all other R groups=H);NMe-C-PhOHDIM (R₁/R_(1′)=CH₃; R₈=C₆H₄OH; all other R groups=H);NMe-C-NaphthylDIM (R₁/R_(1′=CH) ₃; R₈=C₁₀H₇ (naphthyl); all other Rgroups=H (FIG. 7); NMe-C-BiphDIM (R₁/R_(1′)=CH₃; R₈=C₆H₄-C₆H₅; all otherR groups=H); NMe-C-PhMeDIM (R₁/R_(1′)=CH₃; R8=C₆H4-CH₃; other Rgroups=H); NMe-C-Ph-CF₃DIM (R₁/R_(1′)=CH₃; R₈=C₆H₄CF₃; other Rgroups=H). All of these studies were carried out in DMBA-induced rats asdescribed (McDougal et al., “Inhibition of carcinogen-induced ratmammary tumor growth and other estrogen-dependent responses bysymmetrical dihalo-substituted analogs of diindolylmethane” CancerLetts., 151:169-179, 2000) and at doses of 1 or 5 mg/kg every secondday.

[0071] The inventors hypothesize that DIM and related compounds may benot only chemopreventative, but also act as antitumorigenic agents forbladder and other cancers through the AhR and possibly other mechanisticpathways.

[0072] Initial studies on the possible protective role for DIM compoundsin bladder cancer used TCCSUP and J82 human bladder cancer cell linesthat were maintained in RPMI culture media supplemented with 10% fetalbovine serum. Cell proliferation studies were carried out in multi-wellplates, and compounds (in DMSO, 0.1% vol./vol.) were added and cellgrowth was determined over a period of 4-10 days. The growth inhibitoryproperties of DIM and related analogs were determined using thefollowing prototypical compounds: DIM, 5,5′-dimethylDIM (5-Me-DIM),2,2′-dimethylDIM (2-Me-DIM), and 5,5′-dichloroDIM (5-Cl-DIM) atconcentrations from 0.1-10 μM. Proliferation of both human bladdercancer cell lines was significantly inhibited by all compounds used inthis study; moreover, the more sensitive TCCSUP cells were inhibited byall compounds at the lowest concentration (0.1 μM) (FIG. 9).Interestingly, the DIM analogs were more inhibitory than genistein andrelated bioflavonoids in soy products that inhibited growth of thesebladder cancer cells at 10 μM concentrations. In vivo studies show thatgenistein and related soy products inhibit mouse bladder carcinogenesis(Zhou et al.,“Inhibition of murine bladder tumorigenesis by soyisoflavones via alterations in the cell cycle, apoptosis andangiogenesis” Cancer Res., 58:5231-5238, 1998) suggesting that the DIMswill also be antitumorigenic for bladder cancer. These results suggestthat DIM analogs exhibit excellent potential as inhibitors of bladdercancer since the soy products were also active as in vivo inhibitors ofbladder cancer tumor growth.

[0073] Preliminary studies with HT-29 human colon cancer cells alsoindicate that DIM and related analogs protect against colon cancer.HT-29 cells were grown in DMEM and serum and treated with 25 μM DIM and4,4′-dichloroDIM. After 72 hours, there was a 73 and 92% decrease incell proliferation, respectively, and these antiproliferative effectscould be observed with concentrations as low as 10 μM. These growthinhibitory effects were also accompanied by programmed cell death, orapoptosis.

[0074] These cell culture and in vivo results demonstrate that DIMcompounds also inhibit growth of multiple tumor cells indicating abroader application for the DIM compounds in cancer chemotherapy.

[0075] The inventors previously described the combined use of Ahreceptor-based alkyl PCDFs with clinically-used ER antagonists such astamoxifen, and contemplate that in the present disclosure the use ofboth C- and ring-substituted DIMs in combination treatment with ERantagonists such as tamoxifen may provide one example of a combinedtherapy approach to the treatment of breast cancer. Previous studieshave demonstrated that DIM and ring-substituted DIM analogs exhibitantiestrogenic activities in breast cancer cells and antitumorigenicactivity in the carcinogen-induced rat mammary tumor model. However, theactivities of the C-substituted analogs have not been determined.Moreover, it might be expected that C-substituted with alkyl, phenyl orother aromatic substituents would decrease binding affinity to the Ahreceptor and render these compounds inactive as Ah receptor-basedantiestrogens.

[0076] Several studies have previously demonstrated that AhR agonistsexhibit antiestrogenic activity in both in vivo and in vitro models.Research in this laboratory has identified a series of alternatesubstituted alkyl polychlorinated dibenzofurans (PCDFs) which bind tothe AhR, exhibit low toxicity but are relatively potent antiestrogens inboth in vivo and in vitro studies. These compounds inhibit mammary tumorgrowth in female Sprague-Dawley rats initiated with7,12-dimethylbenz[a]-anthracene (DMBA) and the antiturnorigenicactivities of alkyl PCDFs are not accompanied by any apparent liver orextrahepatic toxic effects. Comparable studies have been carried outusing DIM and ring-substituted DIMs (patent application pending) andthese compounds are also relatively nontoxic Ah receptor-basedantiestrogens that block mammary tumor growth in vivo.

[0077] Surprisingly, the C-substituted DIMs retained their bindingaffinity for the Ah receptor and IC₅₀ competitive binding values of1.1×10⁻⁸,9.8×10⁻⁸,5.5×10⁻¹⁰, and 1.8×10⁻⁹ M have been observed for theC-methyl, C-phenyl, 1-methyl/C-p-chlorophenyl and 1-methyl/C-biphenylsubstituents, respectively.

[0078] In addition, it has been demonstrated that several C-substitutedDIMs exhibit antiestrogenic activity in both T47D and MCF-7 human breastcancer cells. The results in FIG. 1 show that C-phenyl and C-methylDIMalone did not induce breast cancer cell proliferation whereas incombination with 1 nM of E2, the hormone-induced proliferative responsewas inhibited.

[0079]FIG. 2 illustrates the potent inhibition of mammary tumor growthby C-phenylDIM in DMBA-induced Sprague-Dawley rats. The anticarcinogenicresponse was observed at a dose as low as 1.0 mg/kg every 2 days, and,as shown in Table 1, this was not accompanied by changes in body/organweights on altered tissue histopathology.

[0080] The inventors have previously shown the utility and advantages ofcombined treatment with Ah receptor-based alkylPCDFs plus tamoxifen andother ER antagonists in the treatment of breast cancer. Both ring- andC-substituted DIMs (see FIG. 10) are also Ah receptor-basedantiestrogens and exhibit antiestrogenic activity in both the breast anduterus, and thus these compounds in combination with ER antagonists suchas tamoxifen can also be used in combined therapy. The combined therapywould act synergistically or additively in blocking tumor growth in thebreast and also protect against potential induction of endometrialcancer by ER antagonists such as tamoxifen.

[0081] All of the compositions and/or methods disclosed and claimedherein can be made and executed without undue experimentation in lightof the present disclosure. While the compositions and methods of thisinvention have been described in terms of preferred embodiments, it willbe apparent to those of skill in the art that variations may be appliedto the compositions and/or methods and in the steps or in the sequenceof steps of the methods described herein without departing from theconcept, spirit and scope of the invention. More specifically, it willbe apparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention.

1.-70. (canceled)
 71. A chemical compound of the formula:

wherein: R₁, R₂, R₄, R₅, R₆, R₇, R₁′, R₂′, R₄′, R₅′, R₆′, and R₇′ areeach independently selected from the group consisting of hydrogen, ahalogen, a linear alkyl group containing one to about ten carbon atoms,a branched alkyl group containing one to about ten carbon atoms, analkoxy group containing one to about ten carbon atoms, and a nitrogroup; at least one of R₁, R₂, R₄, R₅, R₆, R₇, R₁′, R₂′, R₄′, R₅′R₆′,and R₇′ are not hydrogen and at least one of R₈ and R₈′ is not hydrogen.72. A chemical compound of the formula:

wherein: R₁, R₂, R₄, R₅, R₆, R₇, R₁′, R₂′, R₄′, R₅′, R₆′ and R₇′ areeach independently selected from the group consisting of hydrogen, ahalogen, a linear alkyl group containing one to about ten carbon atoms,a branched alkyl group containing one to about ten carbon atoms, analkoxy group containing one to about ten carbon atoms, and a nitrogroup; at least one of R₁, R₂, R₄, R₅, R₆, R₇, R₁′, R₂′, R₄′, R₅′, R₆′,and R₇′ are not hydrogen R₈ and R₈′ are each independently selected fromthe group consisting of hydrogen, a linear alkyl group containing one toabout ten carbon atoms, a branched alkyl group containing one to aboutten carbon atoms, a cycloalkyl group containing one to about ten carbonatoms, and an aryl group; and at least one of R₈ and R₈′ is nothydrogen.
 73. The chemical compound of claim 72, wherein the halogen ischlorine, bromine, or fluorine.
 74. The chemical compound of claim 72,wherein: R₁, R₂, R₄, R₆, R₇, R₁′, R₂′, R₄′, R₆′, and R₇′ are eachhydrogen; and R₅ and R₅′ are each individually a halogen.
 75. Thechemical compound of claim 72, wherein: R₁, R₂, R₄, R₆, R₇, R₁′, R₂′,R₄′, R₆′, and R₇′ are each hydrogen; and R₅ and R₅′ are eachindividually a linear alkyl group.
 76. The chemical compound of claim72, wherein: R₁, R₂, R₄, R₆, R₇, R₁′, R₂′, R₄′, R₆′, and R₇′ are eachhydrogen; and R₅ and R₅′ are each individually an alkoxy group.
 77. Thechemical compound of claim 72, wherein: R₂, R₄, R₅, R₆, R₇, R₂′, R₄′,R₅′, R₆′, and R₇′ are each hydrogen; and R₁ and R₁′ are eachindividually an alkyl group.
 78. The chemical compound of claim 72,wherein: R₂, R₄, R₅, R₆, R₇, R₂′, R₄′, R₅′, R₆′, and R₇′ are eachhydrogen; and R₁ and R₁′ are each individually an alkoxy group.
 79. Thechemical compound of claim 72, wherein: R₁, R₄, R₅, R₆, R₇, R₁′, R₄′,R₅′, R₆′, and R₇′ are each hydrogen; and R₂ and R₂′ are eachindividually an alkyl group.
 80. The chemical compound of claim 72,wherein: R₁, R₂, R₄, R₆, R₇, R₁′, R₂′, R₄′, R₆′, and R₇′ are hydrogen;and R₅ and R₅′ are each a nitro group.
 81. The chemical compound ofclaim 72, wherein at least one of R₈ and R₈′ is a linear alkyl group.82. The chemical compound of claim 72, wherein at least one of R₈ andR₈′ is a branched alkyl group.
 83. The chemical compound of claim 72,wherein at least one of R₈ and R₈′ is a cycloalkyl group.
 84. Thechemical compound of claim 72, wherein at least one of R₈ and R₈′ is anaryl group.
 85. The chemical compound of claim 72, wherein: R₁, R₂, R₁′,and R₂′ are each individually hydrogen or methyl; R_(4, R) ₅, R₆, R₇,R₄′, R₅′, R₆′, and R₇′ are each hydrogen; and R₈ and R₈′ are eachindividually hydrogen, methyl, or an aryl group selected from the groupconsisting of C₆H₅, C₆H₄OH, C₆H₄CH₃, C₆H₄CF₃, C₁₀H₇, C₆H₄H₅, andC₆H₄OCH₃.
 86. The chemical compound of claim 72, wherein: R₁, R₂, R₄,R₅, R₆, R₇, R₁′, R₂′, R₄′, R₅′, R₆′ and R₈′ are each hydrogen; and R₈ isthe aryl group p-C₆H₄C₆H₅.
 87. The chemical compound of claim 72,wherein: R₁, R₂, R₄, R₅, R₆, R₇, R₁′, R₂′, R₄′, R₅′, R₆′ and R₈′ areeach hydrogen; and R₈ is the aryl group p-C₆H₄CF₃.
 88. A compositioncomprising a chemical compound of the formula:

wherein: R₁, R₂, R₄, R₅, R₆, R₇, R₁′, R₂′, R₄′, R₅′, R₆′ are eachindependently selected from the group consisting of hydrogen, a halogen,a linear alkyl group containing one to about ten carbon atoms, abranched alkyl group containing one to about ten carbon atoms, an alkoxygroup containing one to about ten carbon atoms, and a nitro group; atleast one of R₁, R₂, R₄, R₅, R₆, R₇, R₁′, R₂′, R₄′, R₅′R₆′, and R₇′ areand at least one of R₈ and R₈′ is not hydrogen.
 89. A compositioncomprising a chemical compound of the formula:

wherein: R₁, R₂, R₄, R₅, R₆, R₇, R₁′, R₂′, R₄′, R₅′, R₆′ are eachindependently selected from the group consisting of hydrogen, a halogen,a linear alkyl group containing one to about ten carbon atoms, abranched alkyl group containing one to about ten carbon atoms, an alkoxygroup containing one to about ten carbon atoms, and a nitro group; atleast one of R₁, R₂, R₄, R₅, R₆, R₇, R₁′, R₂′, R₄′, R₅′ are not hydrogenR₈ and R₈′ are each independently selected from the group consisting ofhydrogen, a linear alkyl group containing one to about ten carbon atoms,a branched alkyl group containing one to about ten carbon atoms, acycloalkyl group containing one to about ten carbon atoms, and an arylgroup; and at least one of R₈ and R₈′ is not hydrogen.
 90. Thecomposition of claim 89, wherein the halogen is chlorine, bromine, orfluorine.
 91. The composition of claim 89, wherein: R₁, R₂, R₄, R₆, R₇,R₁′, R₂′, R₄′, R₆′, and R₇′ are each hydrogen; and R₅ and R₅′ are eachindividually a halogen.
 92. The composition of claim 89, wherein: R₁,R₂, R₄, R₆, R₇, R₁′, R₂′, R₄′, R₆′, and R₇′ are each hydrogen; and R₅and R₅′ are each individually a linear alkyl group.
 93. The compositionof claim 89, wherein: R₁, R₂, R₄, R₆, R₇, R₁′, R₂′, R₄′, R₆′, and R₇′are each hydrogen; and R₅ and R₅′ are each individually an alkoxy group.94. The composition of claim 89, wherein: R₂, R₄, R₅, R₆, R₇, R₂′, R₄′,R₅′, R₆′, and R₇′ are each hydrogen; and R₁ and R₁′are each individuallyan alkyl group.
 95. The composition of claim 89, wherein: R₂, R₄, R₅,R₆, R₇, R₂′, R₄′, R₅′, R₆′, and R₇′ are each hydrogen; and R₁ and R₁′are each individually an alkoxy group.
 96. The composition of claim 89,wherein: R₁, R₄, R₅, R₆, R₇, R₁′, R₄′, R₅′, R₆′, and R₇′ are eachhydrogen; and R₂ and R₂′ are each individually an alkyl group.
 97. Thecomposition of claim 89, wherein: R₁, R₂, R₄, R₆, R₇, R₁′, R₂′, R₄′,R₆′, and R₇′ are hydrogen; and R₅ and R₅′are each a nitro group.
 98. Thecomposition of claim 89, wherein at least one of R₈ and R₈′ is a linearalkyl group.
 99. The composition of claim 89, wherein at least one of R₈and R₈′ is a branched alkyl group.
 100. The composition of claim 89,wherein at least one of R₈ and R₈′ is a cycloalkyl group.
 101. Thecomposition of claim 89, wherein at least one of R₈ and R₈′ is an arylgroup.
 102. The composition of claim 89, wherein: R₁, R₂, R₁′, and R₂′are each individually hydrogen or methyl; R₄, R₅, R₆, R₇, R₄′, R₅′, R₆′,and R₇′ are each hydrogen; and R₈ and R₈′ are each individuallyhydrogen, methyl, or an aryl group selected from the group consisting ofC₆H₅, C₆H₄OH, C₆H₄CH₃, C₆H₄CF₃, C₁₀H₇, C₆H₄C₆H₅, and C₆H₄OCH₃.
 103. Thecomposition of claim 89, wherein: R₁, R₂, R₄, R₅, R₆, R₇, R′, R₂′, R₄′,R₅′, R₆′,R₇ ′, and R₈′ are each hydrogen; and R₈ is the aryl groupp-C₆H₄C₆H₅.
 104. The composition of claim 89, wherein: R₁, R₂, , R₅, R₆,R₇, R₁′, R₂′, R₄′, R₅′, R₆′,R₇′, and R₈′ are each hydrogen; R₈ is thearyl group p-C₆H₄CF₃.